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The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that play an important role in reverse cholesterol transport. We examined the effects of inhibitors of the cholesterol absorption (ezetimibe) and synthesis (statins) on expression of these transporters in HepG2 cells and peripheral blood mononuclear cells (PBMCs) of individuals with primary (and nonfamilial) hypercholesterolemia (HC). A total of 48 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) and 23 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg of each/day/4 weeks). Gene expression was examined in statin- or ezetimibe-treated and control HepG2 cells as well as PBMCs using real-time PCR. In PBMCs, statins and ezetimibe downregulated ABCA1 and ABCG1 mRNA expression but did not modulate NR1H2 (LXR-β) and NR1H3 (LXR-α) levels. Positive correlations of ABCA1 with ABCG1 and of NR1H2 with NR1H3 expressions were found in all phases of the treatments. In HepG2 cells, ABCA1 mRNA levels remained unaltered while ABCG1 expression was increased by statin (1.0-10.0 µM) or ezetimibe (5.0 µM) treatments. Atorvastatin upregulated NR1H2 and NR1H3 only at 10.0 µM, meanwhile ezetimibe (1.0-5.0 µM) downregulated NR1H2 but did not change NR1H3 expression. Our findings reveal that lipid-lowering drugs downregulate ABCA1 and ABCG1 mRNA expression in PBMCs of HC individuals and exhibit differential effects on HepG2 cells. Moreover, they indicate that the ABCA1 and ABCG1 transcript levels were not correlated directly to LXR mRNA expression in both cell models treated with lipid-lowering drugs. Show less

GX sPLA(2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids; it has been implicated in inflammatory diseases, including atherosclerosis. To identify a novel role for group X (GX) secretory phospholipase A(2) (sPLA(2)) in modulating ATP binding casette transporter A1 (ABCA1) and ATP binding casette transporter G1 (ABCG1) expression and, therefore, macrophage cholesterol efflux. The overexpression or exogenous addition of GX sPLA(2) significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA(2) deficiency in mouse peritoneal macrophages was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA(2)-overexpressing J774 cells and increased efflux in GX sPLA(2)-deficient mouse peritoneal macrophages. Gene regulation was dependent on GX sPLA(2) catalytic activity, mimicked by arachidonic acid and abrogated when liver X receptor (LXR)α/β expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA(2) suppresses the ability of LXR to transactivate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand-binding domain. GX sPLA(2) modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA(2) may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating the genes critical for cellular cholesterol efflux. Show less

Migration of CD4-positive lymphocytes into the vessel wall is a critical step in atherogenesis. Recent data suggest that CD4-positive lymphocytes express the nuclear transcription factors Liver-X-Receptor (LXR) alpha and beta with an effect of LXR activators on TH1-cytokine release from these cells. However, the role of LXR in lymphocyte migration remains currently unexplored. Therefore, the present study investigated whether LXR activation might modulate chemokine-induced migration of these cells. Stimulation of CD4-positive lymphocytes with SDF-1 leads to a 2.5 +/- 0.8-fold increase in cell migration (P < 0.05; n = 12). Pretreatment of cells with the LXR activator T0901317 reduces this effect in a concentration-dependent manner to a maximal 0.9 +/- 0.4-fold induction at 1 micromol/L T0901317 (P < 0.05 compared to SDF-1-treated cells; n = 12). Similar results were obtained with the LXR activator GW3965. The effect of LXR activators on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity as determined by PI-3 kinase activity assays. Downstream, T0901317 inhibited activation of the small GTPase Rac and phosphorylation of the myosin light chain (MLC). Moreover, LXR activator treatment reduced f-actin formation as well as ICAM3 translocation to the uropod of the cell, thus interfering with two important steps in T cell migration. Transfection of CD4-positive lymphocytes with LXRalpha/beta siRNA abolished T0901317 inhibitory effect on MLC phosphorylation and ICAM3 translocation. LXR activation by T0901317 or GW3965 inhibits chemokine-induced migration of CD4-positive lymphocytes. Given the crucial importance of chemokine-induced T cell migration in early atherogenesis, LXR activators may be promising tools to modulate this effect. Show less

Isoliquiritigenin (ILQ), a flavonoid obtained from Glycyrrhizae species, has an antioxidant effect. This study investigated the potential of ILQ for inhibiting liver X receptor-α (LXRα)-mediated lipogenesis and steatosis in hepatocytes and its underlying molecular basis. Treatment with ILQ antagonized the ability of an LXRα agonist (T0901317) to activate sterol regulatory element binding protein-1c (SREBP-1c), thereby repressing transcription of fatty acid synthase, acetyl-CoA carboxylase, ATP-binding cassette transporter-A1, and stearoyl-CoA desaturase-1. ILQ treatment inhibited activating phosphorylation of JNK1 elicited by palmitate or TNFα. JNK1, but not JNK2, increased LXRα phosphorylation at serine residues, promoting LXRα activation. The ability of ILQ to inhibit JNK1 downstream of ASK1-MKK7 led to the repression of T0901317-inducible LXRα and SREBP-1c activation. In mice fed a high-fat diet, ILQ treatment inhibited hepatic steatosis, as shown by a decrease in fat accumulation and repression of lipogenic genes. The results of blood biochemistry and histopathology confirmed attenuation of high-fat diet-induced liver injury by ILQ. Moreover, ILQ inhibited oxidative stress, as indicated by decreases in thiobarbituric acid-reactive substance formation, iNOS and COX2 induction, and nitrotyrosinylation. Our results demonstrate that ILQ has the ability to repress LXRα-dependent hepatic steatosis through JNK1 inhibition and protect hepatocytes from oxidative injury inflicted by fat accumulation. Show less

The liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily that function as key transcriptional regulators of a number of biological processes, including cholesterol homeostasis, lipid metabolism, and keratinocyte differentiation. Natural ligands that activate LXRs include oxysterol derivatives such as 25-hydroxycholesterol, 27-hydroxycholesterol, 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Related oxysterols, such as 5α,6α-epoxycholesterol (5,6-EC) are present in a number of foods and have been shown to induce atherosclerosis in animal models. Intriguingly, these oxysterols have also been detected in atherosclerotic plaques. Using a variety of biochemical and cellular assays, we demonstrate that 5,6-EC is the first dietary modulator and an endogenous LXR ligand with cell and gene context-dependent antagonist, agonist, and inverse agonist activities. In a multiplexed LXR-cofactor peptide interaction assay, 5,6-EC induced the recruitment of a number of cofactor peptides onto both LXRα and LXRβ and showed an EC(50) of approximately 2 μM in peptide recruitment. Furthermore, 5,6-EC bound to LXRα in a radiolabeled ligand displacement assay (EC(50) = 76 nM), thus demonstrating it to be one of the most potent natural LXRα ligands known to date. Analysis of endogenous gene expression in various cell-based systems indicated the potential of 5,6-EC to antagonize LXR-mediated gene expression. Furthermore, it also induced the expression of some LXR-responsive genes in keratinocytes. These results clearly demonstrate that 5,6-EC is an LXR modulator that may play a role in the development of lipid disorders, such as atherosclerosis, by antagonizing the agonistic action of endogenous LXR ligands. Show less

Transcription factors and nuclear receptors constitute a link between exposure to heterocyclic amines and polycyclic aromatic hydrocarbons from meat and tobacco smoke and colorectal cancer (CRC) risk. The aim of this study was to investigate if polymorphisms in nuclear factor kappa-B, pregnane X receptor, and liver X receptor were associated with risk of CRC, and to investigate possible interactions with lifestyle factors such as smoking, meat consumption, and NSAID use. The polymorphisms nuclear factor kappa-B (NFkB, NFKB1) -94 insertion/deletion ATTG (rs28362491), pregnane X receptor (PXR, NR1I2) A-24381C (rs1523127), C8055T (rs2276707), A7635G (rs6785049), liver X receptor (LXR-β, NR1H3) C-rs1405655T, T-rs2695121C were assessed together with lifestyle factors in a nested case-cohort study of 378 CRC cases and 756 random participants from the Danish prospective Diet, Cancer and Health study of 57,053 persons. Carriers of NFkB -94deletion were at 1.45-fold higher risk of CRC than homozygous carriers of the insertion allele (incidence rate ratio (IRR) = 1.45, 95% confidence interval (95% CI): 1.10-1.92). There was interaction between this polymorphism and intake of red and processed meat in relation to CRC risk. Carriers of NFkB -94deletion were at 3% increased risk pr 25 gram meat per day (95% CI: 0.98-1.09) whereas homozygous carriers of the insertion were not at increased risk (p for interaction = 0.03). PXR and LXR polymorphisms were not associated with CRC risk. There was no interaction between use of nonsteroid antiinflammatory drugs (NSAID) or smoking status and NFkB, PXR or LXR polymorphisms. A polymorphism in NFkB was associated with CRC risk and there was interaction between this polymorphism and meat intake in relation to CRC risk. This study suggests a role for NFkB in CRC aetiology. Show less

The metabolic syndrome constitutes a group of metabolic conditions that increase the risk of developing diseases, including cardiovascular disease (CVD) and type 2 diabetes (T2D). LXRα/β are regulators of lipogenesis, cholesterol/glucose homoeostasis and inflammatory pathways, processes that are intertwined with development of the metabolic syndrome. The employment of LXRs as pharmaceutical targets for treatment of various aspects of the metabolic syndrome has been promptly investigated but serious side effects, like hepatic steatosis, have hampered this process. Novel treatment regimes now focus on development of isoform-specific or tissue-specific LXR agonist/antagonist compounds to circumvent effects on lipid biosynthesis. Other strategies to explore the beneficial aspects of LXR activation include targeting co-factors or pathways that are modifying LXR activity. Show less

Organic anion transporting polypeptide 1B1 (OATP1B1) is a liver-enriched transporter involved in the hepatocellular uptake of many endogenous molecules and several structurally divergent drugs in clinical use. Although OATP1B1 coding region polymorphisms are known to make an impact on substrate drug disposition in humans, little is known regarding the mechanisms underlying the transcriptional regulation of this transporter. In this study, we note that messenger RNA (mRNA) expression of OATP1B1 in a large human liver bank exhibited marked interindividual variability that was not associated with coding region polymorphisms. Accordingly, we hypothesized that such variability in expression is reflective of nuclear receptor-mediated transcriptional regulation of this transporter. We tested prototypical ligands for the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) α, and farnesoid X receptor (FXR) in a human hepatoma-derived cell line and noted induction of OATP1B1 mRNA when the cells were treated with LXRα or FXR ligands. To confirm a direct role for LXRα and FXR to OATP1B1 expression, we performed detailed promoter analysis and cell-based reporter gene assays resulting in the identification of two functional FXR response elements and one LXRα response element. The direct interaction between nuclear receptors with the identified response elements was assessed using chromatin immunoprecipitation assays. Using isolated primary human hepatocytes, we show that LXRα or FXR agonists, but not PXR or CAR agonists, are capable of OATP1B1 induction. We note that OATP1B1 transcriptional regulation is under dual nuclear receptor control through the oxysterol sensing LXRα and the bile acid sensor FXR. Accordingly, the interplay between OATP1B1 and nuclear receptors may play an important and heretofore unrecognized role during cholestasis, drug-induced liver injury, and OATP1B1 induction-related drug interactions. Show less

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). PGC-1α enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1α levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1α-mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1α and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1α enhances lipogenesis in skeletal muscle through liver X receptor α-dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1α and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1α coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo. Show less

The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia. We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists. Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes. In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317. LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport. Show less

C-reactive protein (CRP) serves not only as a biomarker for the risk of cardiovascular disease and underlying inflammation but also functions as an active mediator of atherosclerosis by promoting activation of endothelial cells and monocytes. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) transcription factor has been recognized to regulate the expression of many genes involved in inflammation, lipid metabolism and vascular remodeling. Therefore, in the present study we tried to explore the role of CRP as a possible mediator of atherosclerosis by determining its effect on PPAR-gamma and its effector genes, i.e., liver X receptor-alpha (LXR-alpha) and matrix metalloproteinase-9 (MMP-9) in THP-1 cells. Semi-quantitative RT-PCR was used to determine mRNA expression. CRP upregulates the expression of PPAR-gamma and LXR-alpha at lower doses (5-25 microg/mL), which were further declined at higher doses (50-100 microg/mL). However, a dose-dependent increase was observed for MMP-9 expression. Atorvastatin (10-20 microM) was able to significantly accelerate the CRP-induced expression of PPAR-gamma and LXR-alpha and attenuate MMP-9 expression. For the first time we demonstrate that CRP modulates PPAR-gamma and its effector genes and reinforces the mechanistic link of CRP as a possible mediator in atherosclerosis and also advocate atorvastatin as a therapeutic modality. Show less

Gallbladder cancer is a highly aggressive disease with poor prognosis that is two to six times more frequent in women than men. The development of gallbladder cancer occurs over a long time (more than 15 y) and evolves from chronic inflammation to dysplasia/metaplasia, carcinoma in situ, and invasive carcinoma. In the present study we found that, in female mice in which the oxysterol receptor liver X receptor-beta (LXRbeta) has been inactivated, preneoplastic lesions of the gallbladder developed and evolved to cancer in old animals. LXRbeta is a nuclear receptor involved in the control of lipid homeostasis, glucose metabolism, inflammation, proliferation, and CNS development. LXRbeta(-/-) female gallbladders were severely inflamed, with regions of dysplasia and high cell density, hyperchromasia, metaplasia, and adenomas. No abnormalities were evident in male mice, nor in LXRalpha(-/-) or LXRalpha(-/-)beta(-/-) animals of either sex. Interestingly, the elimination of estrogens with ovariectomy prevented development of preneoplastic lesions in LXRbeta(-/-) mice. The etiopathological mechanism seems to involve TGF-beta signaling, as the precancerous lesions were characterized by strong nuclear reactivity of phospho-SMAD-2 and SMAD-4 and loss of E-cadherin expression. Upon ovariectomy, E-cadherin was reexpressed on the cell membranes and immunoreactivity of pSMAD-2 in the nuclei was reduced. These findings suggest that LXRbeta in a complex interplay with estrogens and TGF-beta could play a crucial role in the malignant transformation of the gallbladder epithelium. Show less

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells. Show less

In order to understand the molecular mechanisms underlying effects of feeding rice protein on lipid and glucose homeostasis, weanling rats were fed AIN-93G diets made with casein or rice protein isolate (RPI) for 14 d. Peroxisome proliferator-activated receptor (PPAR)alpha genes and proteins involved in fatty acid degradation were upregulated by feeding RPI (P < 0.05), accompanied by increased promoter binding and nuclear expression of PPARalpha and its heterodimerization partner retinoid X receptor (P < 0.05). Effects of RPI feeding on hepatic PPARgamma signaling were significant but less robust. Feeding RPI also increased hepatic genes involved in cholesterol metabolism and transport. However, feeding RPI had no effect on binding of liver X-receptor (LXR)alpha to the cytochrome P450 (CYP)7A1 promoter. The effect of RPI feeding on PPARalpha signaling appeared to be direct and was reversed when RPI diets were switched to casein. In another experiment, male Sprague-Dawley rats were fed casein diets from postnatal day (PND) 24 to PND64 or were fed high fat 'Western' diets containing 0.5% cholesterol made with either casein or RPI. Increased liver triglyceride content, hypercholesterolemia and insulin resistance in the 'Western' diet-fed rats were partially prevented by feeding RPI (P < 0.05). mRNA and protein expression of hepatic enzymes involved in fatty acid synthesis were suppressed by feeding 'Western diets' containing RPI (P < 0.05), despite a lack of effects on nuclear concentrations of sterol regulatory element binding protein-1c. These data suggest that attenuation of metabolic syndrome observed in RPI-fed rats after consumption of diets high in fat and cholesterol occur as a result of improved lipid and glucose homeostasis partly as a result of activation of PPARs. Show less

Preeclampsia is a pregnancy-specific disorder associated with hyperlipidemia. Liver X receptor (LXR) alpha and LXRbeta are key regulators of lipid homeostasis. In the current study, we investigated expression of LXRalpha, LXRbeta and their target genes in human term placenta, decidua and subcutaneous adipose tissue from pregnancies complicated by preeclampsia. Furthermore, we analyzed the protein levels of LXRalpha and LXRbeta in placenta. We also analyzed lipid concentrations in term placental tissue. Gene expression of LXRalpha, LXRbeta and fatty acid transporter CD36 was significantly decreased in placental tissues, while increased expression was observed for LXRalpha in adipose tissue, from pregnancies complicated by preeclampsia. The placental protein level of LXRbeta was reduced, and there was a positive correlation between placental LXRbeta mRNA expression and placental free fatty acids in preeclampsia. Our results suggest a possible role for LXRbeta as a transcriptional regulator in preeclampsia. Show less

In addition to their role in dietary lipid absorption bile acids are signaling modules activating nuclear receptors and at least one G-protein coupled receptors named the TGR5. With a different rank of potency primary and secondary bile acids activates a subset of nuclear receptors including the farnesoid-X-receptor (FXR, NR1H4); the constitutive androstane receptor (CAR, NR1H3), the pregnane-x- receptor (PXR, NR1H2), the vitamin D receptor (VDR, NR1H1). Originally, these receptors were characterized for their role as bile acid and xenobiotic sensors, emerging evidence, however, indicates that FXR, PXR and VDR and their ligands are important for the modulation of immune and inflammatory reactions in entero-hepatic tissues. The immune phenotype FXR deficient mice indicates that these receptors are essential for the maintenance of immune homeostasis. A common theme of all bile acid-activated receptor is their ability to counter-regulate effector activities of cells of innate immunity establishing that signals generated by these receptors and their ligands function as a braking signals for inflammation in entero-hepatic tissues. In this review, we will spotlight the molecular mechanisms of receptor/ligand function and how bile acid-activated receptors regulate the innate immunity in the gastrointestinal tract and liver. The ability of these receptors to integrate metabolic and inflammatory signaling makes them particularly attractive targets for intervention in immune-mediated diseases. Show less

First discovered as orphan receptors, liver X receptors (LXRs) were subsequently identified as the nuclear receptor target of the cholesterol metabolites, oxysterols. There are 2 LXR receptors encoded by distinct genes: LXRalpha is most highly expressed in the liver, adipose, kidney, adrenal tissues, and macrophages and LXRbeta is ubiquitously expressed. Despite differential tissue distribution, these isoforms have 78% homology in their ligand-binding domain and appear to respond to the same endogenous ligands. Work over the past 10 years has shown that the LXR pathway regulates lipid metabolism and inflammation via both the induction and repression of target genes. Given the importance of cholesterol regulation and inflammation in the development of cardiovascular disease, it is not surprising that activation of the LXR pathway attenuates various mechanisms underlying atherosclerotic plaque development. In this brief review, we will discuss the impact of the LXR pathway on both cholesterol metabolism and atherosclerosis. Show less

Liver X receptor (LXR) is a nuclear receptor regulating cholesterol metabolism. Liver X receptor has also been shown to exert anti-proliferative and anti-inflammatory properties. In this study, we evaluated the effect of LXR activation on cardiac hypertrophy in vitro and in vivo. Treatment with the synthetic LXR agonist T0901317 (T09) attenuated the hypertrophic response of cultured cardiomyocytes to endothelin-1 almost to control levels. siRNA interference showed that this effect was indeed LXR specific. To corroborate these findings in vivo, abdominal aortic constriction (AC) was used as a pressure overload model to induce cardiac hypertrophy in wild-type and LXR-α-deficient (LXR-α(-/-)) mice. In wild-type mice, T09 treatment resulted in a decrease of cardiac wall thickening 4 and 7 weeks after AC. Also, after 7 weeks of AC, mean arterial blood pressure and left ventricular weight/body weight (LVW/BW) ratios were decreased in T09 treated mice. These effects were not observed in LXR-α(-/-) mice, indicating that the beneficial effect of LXR activation on cardiac hypertrophy is attributable to the LXR-α isoform. T09 induced robust cardiac expression of metabolic genes which are downstream of LXR-α, such as SREBP-1c, ABCA1, and ABCG1. Together these results indicate that LXR exerts salutary effects in cardiac hypertrophy, possibly via metabolic remodelling. Show less

Berberine, a botanical alkaloid purified from Cortidis rhizoma, has effects in cardiovascular diseases, yet the mechanism is not fully understood. Foam cells play a critical role in the progression of atherosclerosis. This study aimed to investigate the effect of berberine on the formation of foam cells by macrophages and the underlying mechanism. Treatment with berberine markedly suppressed oxidized low-density lipoprotein (oxLDL)-mediated lipid accumulation, which was due to an increase in cholesterol efflux. Berberine enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Additionally, functional inhibition of ABCA1 with a pharmacological inhibitor or neutralizing antibody abrogated the effects of berberine on cholesterol efflux and lipid accumulation. Moreover, berberine induced the nuclear translocation and activation of liver X receptor alpha (LXRalpha) but not its protein expression. Knockdown of LXRalpha mRNA expression by small interfering RNA abolished the berberine-mediated protective effects on ABCA1 protein expression and oxLDL-induced lipid accumulation in macrophages. These data suggest that berberine abrogates the formation of foam cells by macrophages by enhancing LXRalpha-ABCA1-dependent cholesterol efflux. Show less

Liver X receptors (LXRs), LXRalpha and LXRbeta, are members of the nuclear receptor superfamily and regulate the expression of genes involved in the regulation of cholesterol and fatty acid metabolism. Human plasma, unlike mouse plasma, contains cholesteryl ester transfer protein (CETP), which plays an important role in reverse cholesterol transport (RCT). LXRs induce CETP transcription via a direct repeat 4 element in the CETP promoter. However, the specific roles of the individual LXR subtypes in CETP expression and their consequences on plasma lipoprotein metabolism are still unclear. Here we showed that synthetic LXR agonist enhanced plasma CETP activity and resulted in non-high density lipoprotein (non-HDL) increase and HDL decrease in cynomolgus monkeys and human CETP transgenic mice. To address the relative importance of the two LXR subtypes, we investigated the effect of the suppression of both LXR subtypes on CETP expression in HepG2 cells. CETP expression induced by the LXR agonist was significantly reduced by LXRalpha knock-down, but not by LXRbeta. Consistent with these data, CETP promoter activity was enhanced by LXRalpha activation, whereas LXRbeta activation had only a minor effect. Furthermore, we investigated the effect of genetic deficiency of both LXR subtypes in human CETP transgenic mice. LXRalpha deficiency abolished the augmentation of plasma CETP activity and hepatic CETP expression induced by the synthetic LXR agonist, consequently increasing HDL and decreasing non-HDL, whereas LXRbeta deficiency did not affect CETP activation. These findings indicate that LXRalpha has an essential role in the regulation of CETP expression and maintaining RCT. Show less

The mechanisms of action of vitamin A deficiency (VAD) on lipid metabolism in the rat kidney were investigated in adult female rats and their offspring. The rats were randomized into three groups: (1) control, in which the mother and offspring received a normal diet (4000 retinol IU/kg diet) for 8 weeks; (2) VAD group, in which the mothers and offspring received a VAD diet (400 retinol IU/kg diet) for 8 weeks; (3) vitamin A-refed group, in which a group of pups on a VAD diet for 8 weeks received a complete diet (6500 retinol IU/kg diet) for 15 days. The lipid metabolism of the offsprings' kidneys and its relation to the expression of apolipoprotein B100 (Apo-B100), liver X receptor alpha (LXRalpha), and retinoid X receptor-alpha/beta (RXRalpha/beta) mRNA was analyzed. VAD was found to alter renal lipid metabolism and its immune environment due to the expression of Apo-B100. Compared with the control, VAD rats had significantly higher levels of transforming growth factor-beta 1 and lower levels of ABCA1, a key gene involved in cholesterol efflux and tissue lipid homeostasis. The expression of LXRalpha and RXRalpha/beta mRNA also decreased in the VAD rat kidney. Vitamin A refeeding reversed all of the changes. Lipid metabolism involved in renal reverse cholesterol transport may be mediated by decreasing the signaling through the ABCA1 cholesterol efflux pathway, which is significantly modified in kidneys of vitamin A-deficient rats. Show less

Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. To determine whether fatty acid synthesis affects atherosclerosis, we inactivated fatty-acid synthase (FAS) in macrophages of apoE-deficient mice. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals. Atherosclerotic extent was decreased 20-40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor alpha (LXRalpha; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor alpha (PPARalpha) target gene expression was decreased in FASKOM macrophages. PPARalpha agonist treatment of FASKOM and wild type macrophages normalized PPARalpha target gene expression as well as Nr1h3 (LXRalpha). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRalpha-deficient/FAS-deficient bone marrow as compared with LXRalpha-replete/FAS-deficient marrow, consistent with anti-atherogenic effects of LXRalpha in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRalpha and suggest that FAS, which is induced by LXRalpha, may generate regulatory lipids that cause feedback inhibition of LXRalpha in macrophages. Show less

Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species. Show less

Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) and liver X receptor alpha (LXRalpha) are nuclear receptors that play pivotal roles in macrophage cholesterol homeostasis and inflammation; key biological processes in atherogenesis. The activation of PPARgamma1 and LXRalpha by natural or synthetic ligands results in the transactivation of ABCA1, ABCG1, and ApoE; integral players in cholesterol efflux and reverse cholesterol transport. In this review, we describe the structure, isoforms, expression pattern, and functional specificity of PPARs and LXRs. Control of PPARs and LXRs transcriptional activity by coactivators and corepressors is also highlighted. The specific roles that PPARgamma1 and LXRalpha play in inducing macrophage cholesterol efflux mediators and antagonizing macrophage inflammatory responsiveness are summarized. Finally, this review focuses on the recently reported regulatory functions that adipocyte enhancer-binding protein 1 (AEBP1) exerts on PPARgamma1 and LXRalpha transcriptional activity in the context of macrophage cholesterol homeostasis and inflammation. Show less

Liver X receptors (LXRs) promote macrophage reverse cholesterol transport and cholesterol excretion from the body. The synthetic LXR ligands T0901317 and GW3965 were shown to significantly inhibit atherosclerosis in mice and to increase the expression of ATP-binding cassette transporter A1 (ABCA1) in the atherosclerotic lesions. However, these compounds increase plasma and hepatic triglyceride (TG) levels in mice. Methyl-3β-hydroxy-5α,6α-epoxycholanate (MHEC), synthesized from hyodeoxycholic acid, functions as an LXR agonist, but its role in atherogenesis and lipid metabolism remained to be elucidated. THP-1-derived macrophages were cultured in the medium con- taining various concentrations of MHEC or T0901317 (0-10 μmol/l) for 24 h. Reverse transcription polymerase chain reaction was used to quantify LXRα, LXRβ and ABCA1 mRNA levels in macrophages. Additionally, MHEC or T0901317 was orally administered at 10 mg/kg daily for 6 weeks in apolipoprotein E knockout (apoE⁻/⁻) mice fed a high-cholesterol diet. Plasma lipids were determined enzymatically. The area of and ABCA1 expression in the aortic atherosclerotic lesions were measured by oil red O staining and immunohistochemistry, respectively. Both MHEC and T0901317 equally stimulated LXRα and ABCA1 mRNA expression in a dose-dependent manner in THP-1-derived macrophages, but they did not induce LXRβ mRNA expression significantly. The plasma levels of total cholesterol, TG and high-density lipoprotein cholesterol were significantly higher in T0901317-treated mice than in the vehicle-treated control group. Interestingly, MHEC treatment dramatically increased plasma high-density lipoprotein cholesterol without altering plasma levels of total cholesterol and TG. Both MHEC and T0901317 equally inhibited the development of atherosclerotic lesions in apoE⁻/⁻ mice. The expression of ABCA1, a cholesterol efflux transporter, was greatly induced by the two LXR agonists in the artery wall. MHEC is a novel LXR agonist and it inhibits atherosclerosis in apoE⁻/⁻ mice without raising blood TG. Thus, MHEC relative to T0901317 may be a better therapeutic LXR agonist for the treatment of atherosclerosis. Show less

The liver X receptors LXRalpha and LXRbeta play critical roles in maintaining lipid homeostasis by functioning as transcription factors that regulate genetic networks controlling the transport, catabolism, and excretion of cholesterol. The studies described in this report examine the individual anti-atherogenic activity of LXRalpha and LXRbeta and determine the ability of each subtype to mediate the biological response to LXR agonists. Utilizing individual knockouts of LXRalpha and LXRbeta in the Ldlr(-/-) background, we demonstrate that LXRalpha has a dominant role in limiting atherosclerosis in vivo. Functional studies in macrophages indicate that LXRalpha is required for a robust response to LXR ligands, whereas LXRbeta functions more strongly as a repressor. Furthermore, selective knockout of LXRalpha in hematopoietic cells and rescue experiments indicate that the anti-atherogenic activity of this LXR subtype is not restricted to macrophages. These studies indicate that LXRalpha plays a selective role in limiting atherosclerosis in response to hyperlipidemia. Show less

In addition to the hematopoietic effect of erythropoietin, increasing evidence suggests that erythropoietin also exerts protective effects for cardiovascular diseases. However, the role of erythropoietin and its underlying mechanism in macrophage foam cell formation are poorly understood. Compared with wild-type specimens, erythropoietin was increased in atherosclerotic aortas of apolipoprotein E-deficient (apoE(-/-)) mice, mainly in the macrophage foam cells of the lesions. Erythropoietin levels in culture medium and macrophages were significantly elevated in response to oxidized low-density lipoprotein in a dose-dependent manner. Furthermore, erythropoietin markedly attenuated lipid accumulation in oxidized low-density lipoprotein-treated macrophages, a result that was due to an increase in cholesterol efflux. Erythropoietin treatment significantly increased ATP-binding cassette transporters (ABC) A1 and ABCG1 mRNA and protein levels without affecting protein expression of scavenger receptors, including scavenger receptor-A, CD36, and scavenger receptor-BI. The upregulation of ABCA1 and ABCG1 by erythropoietin resulted from liver X receptor alpha activation, which was confirmed by its prevention on expression of ABCA1 and ABCG1 after pharmacological or small interfering RNA inhibition of liver X receptor alpha. Moreover, the erythropoietin-mediated attenuation on lipid accumulation was abolished by such inhibition. Finally, reduced lipid accumulation and marked increase in ABCA1 and ABCG1 were demonstrated in erythropoietin-overexpressed macrophages. Our data suggest that erythropoietin suppresses foam cell formation via the liver X receptor alpha-dependent upregulation of ABCA1 and ABCG1. Show less

Liver X receptor (LXR) α and β are nuclear receptors that control cellular metabolism. LXRs modulate the expression of genes involved in cholesterol and lipid metabolism in response to changes in cellular cholesterol status. Because of their involvement in cholesterol homeostasis, LXRs have emerged as promising drug targets for anti-atherosclerotic therapies. In rodents, synthetic LXR agonists promote cellular cholesterol efflux, transport and excretion. As a result, the progression of atherosclerosis is halted. However, pharmacological LXR activation also induces hepatic steatosis and promotes the secretion of atherogenic triacylglycerol-rich VLDL particles by the liver, complicating the clinical application of LXR agonists. The more recently emerged roles of LXRs in fat tissue, pituitary and brain may have implications for treatment of obesity and Alzheimer disease. In addition to the improvements in atherosclerosis, LXR activation exerts beneficial effects on glucose control in mouse models of type 2 diabetes. Future therapeutic strategies aiming to exert beneficial effects on cholesterol and glucose homeostasis, while circumventing the undesired effects on hepatic lipid metabolism, should target specific LXR-mediated processes. Therefore, tissue and/or isotype-specific effects of LXR action need to be established. The consequences of combinatorial drug approaches and the identification of the co-regulatory networks involved in the LXR-mediated control of particular genes may contribute to development of novel LXR agonists. Finally, pathway analyses of LXR actions provide tools to evaluate and optimize the effectiveness of novel therapeutic strategies to prevent and/or treat metabolic diseases. Show less

Liver X receptor (LXR) alpha and beta are ligand-activated transcription factors that regulate the expression of genes involved in the removal of cholesterol from cells by high-density lipoproteins, the transport of cholesterol to the liver and the biliary excretion of cholesterol. LXRs are activated by oxygenated cholesterol derivatives such as 24(S),25-epoxycholesterol or 24(S)-, 25- and 27-hydroxycholesterol. In this review, we will discuss the role of LXR in the reproductive system and perspectives on the application of LXR agonists in the treatment of reproductive pathologies. Interestingly, progressive age-related impairment of fertility is observed in both female and male LXR knockout mice. Reduced fertility in female LXR knockout mice is associated with resistance to follicular fluid meiosis-activating sterol (FF-MAS), the intermediate of cholesterol synthesis generated in the ovaries that is responsible for stimulating oocyte meiosis partially in a LXR-dependent manner. Female mice lacking both LXR isoforms exhibit symptoms of ovarian hyperstimulation syndrome when treated with pharmacological doses of gonadotropins. LXR agonists have mainly been considered as potential anti-atherosclerotic medications. However, experimental studies suggest that natural or synthetic LXR agonists may also effectively treat some reproductive pathologies, such as infertility, impaired uterine contractility, hormone-dependent cancers and insulin resistance in patients with polycystic ovarian syndrome. However, the specific adverse effects of LXR agonists on the reproductive system must also be considered. Adverse effects of LXR agonists include impaired trophoblast invasion, excessive transplacental cholesterol transport from the mother to the fetus leading to fetal hypercholesterolemia, and augmented estrogen deficiency after menopause. Show less

A series of phenyl sulfone substituted quinoxaline were prepared and the lead compound 13 (WYE-672) was shown to be a tissue selective LXR Agonist. Compound 13 demonstrated partial agonism for LXRbeta in kidney HEK-293 cells but did not activate Gal4 LXRbeta fusion proteins in huh-7 liver cells. Although 13 showed potent binding affinity to LXRbeta (IC(50) = 53 nM), it had little binding affinity for LXRalpha (IC(50) > 1.0 microM) and did not recruit any coactivator/corepressor peptides in the LXRalpha multiplex assay. However, compound 13 showed good agonism in THP-1 cells with respect to increasing ABCA1 gene expression and good potency on cholesterol efflux in THP-1 foam cells. In an eight-week lesion study in LDLR -/- mice, compound 13 showed reduction of aortic arch lesion progression and no plasma or hepatic triglyceride increase. These results suggest quinoxaline 13 may have an improved biological profile for potential use as a therapeutic agent. Show less